Arylpropionyl-alpha-pyrone antibacterial agents

ABSTRACT

The invention provides a compound of formula (I): or a salt thereof, wherein R 1 -R 4  have any of the values described in the specification, as well as compositions comprising a compound of formula (I). The compounds are useful as antibacterial agents.

PRIORITY OF INVENTION

This application claims priority to U.S. Provisional Application No.61/381,109, filed 9 Sep. 2010.

GOVERNMENT FUNDING

The invention described herein was made with United States Governmentsupport under Grant Number ROI-AI072766 awarded by the NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The emergence of multidrug-resistant (MDR) bacterial pathogens (e.g.methicillin-resistant Staphylococcus aureus, MRSA) has increasedconcerns as to the adequacy of current antimicrobials and pathogentreatment methods. The lethality of such pathogens has often led totreatment methods that are experimental or would otherwise normally beavoided in standard clinical practice. For example, the antibioticcolistin was traditionally considered too nephrotoxic and neurotoxic forclinical use, but is nevertheless used to treat MDR bacterial infectionsdue to a paucity of available active drugs. The growing threat from MDRpathogens highlights a critical need for new antibiotics that exhibitnovel mechanisms of action and/or that are able to circumvent knownresistance pathways.

SUMMARY OF THE INVENTION

Applicant has identified compounds that inhibit bacterial RNA polymeraseand inhibit bacterial growth. Accordingly, in one embodiment theinvention provides a compound of formula I:

wherein:

R₁ is one of aryl and heteroaryl, and optionally is substituted with oneor more groups independently selected from halo, hydroxy, carboxy,cyano, nitro, trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl,heteroaryl, aryloxy, heteroaryloxy, and —NR^(a)R^(b);

R₂ is one of H, halo, hydroxy, carboxy, cyano, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy, heteroaryloxy, or—NR^(c)R^(d), wherein any (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, andheteroaryl, and is optionally substituted with one or more halo,hydroxy, carboxy, cyano, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy or —NR^(c)R^(d);

R₃ is one of H, halo, hydroxy, carboxy, cyano, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy, heteroaryloxy, or—NR^(e)R^(f), wherein any (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, andheteroaryl, and is optionally substituted with one or more halo,hydroxy, carboxy, cyano, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy or —NR^(e)R^(f);

R₄ is (C₂-C₆)alkyl or (C₂-C₆)alkenyl, which (C₂-C₆)alkyl or(C₂-C₆)alkenyl is optionally substituted with one or more groupsindependently selected from oxo, halo, hydroxy, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy,heteroaryloxy, and —NR^(g)R^(h);

R^(a) and R^(b) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(a) andR^(b) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino;

R^(c) and R^(d) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(c) andR^(d) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino;

R^(e) and R^(f) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(e) andR^(f) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino; and

R^(g) and R^(h) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(g) andR^(h) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino;

or a salt thereof.

The invention also provides a composition comprising a compound offormula I or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable vehicle.

The invention also provides a method for inhibiting a bacterial RNApolymerase comprising contacting the bacterial RNA polymerase with acompound of formula I or a salt thereof.

The invention also provides a method for inhibiting the growth of abacteria comprising contacting the bacteria with a compound of formula Ior a salt thereof.

The invention also provides a method for treating a bacterial infectionin a mammal comprising administering to the mammal an effective amountof a compound of formula I or a pharmaceutically acceptable saltthereof.

The invention also provides a compound of formula I or apharmaceutically acceptable salt thereof for use in the prophylactic ortherapeutic treatment of a bacterial infection.

The invention also provides a compound of formula I or apharmaceutically acceptable salt thereof for use in medical treatment.

The invention also provides the use of a compound of formula I or apharmaceutically acceptable salt thereof for the preparation of amedicament for treating a bacterial infection in a mammal.

The invention also provides the use of a compound of formula I or a saltthereof as a disinfectant, sterilant, antispoilant, or antiseptic.

The invention also provides synthetic processes and intermediatesdisclosed herein that are useful for preparing compounds of formula (I)or salts thereof.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc.denote both straight and branched groups; but reference to an individualradical such as propyl embraces only the straight chain radical, abranched chain isomer such as isopropyl being specifically referred to.Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to ten ring atoms in which at least one ringis aromatic. Heteroaryl encompasses a radical of a monocyclic aromaticring containing five or six ring atoms consisting of carbon and one tofour heteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is H, O, (C₁-C₄)alkyl,phenyl or benzyl, as well as a radical of an ortho-fused bicyclicheterocycle of about eight to ten ring atoms comprising one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X).

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase.

It will also be appreciated that compounds of the invention can exist indifferent tautomeric forms. The compounds of formula I also encompassesany tautomeric forms or mixtures thereof. For example, two possibletautomeric forms of a compound of formula I are illustrated below inScheme 1.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₁-C₆)alkoxycan be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can bevinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, or 5-hexenyl; (C₂-C₆)alkynyl can be ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, or 5-hexynyl; (C₁-C₆)alkanoyl can be acetyl, propanoyl orbutanoyl; (C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkanoyloxy can be formyloxy, acetoxy,propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy;aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl,imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl,isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (orits N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl,isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).

In one specific embodiment of the invention R₁ is phenyl which isoptionally substituted with one or more groups independently selectedfrom halo, hydroxy, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy, heteroaryloxy, and—NR^(a)R^(b).

In one specific embodiment of the invention R₁ is phenyl.

In one specific embodiment of the invention R₂ is H or methyl.

In one specific embodiment of the invention R₃ is H or methyl.

In one specific embodiment of the invention R₄ is n-butyl or i-pentyl.

In one specific embodiment the invention provides a compound of formulaI wherein:

R₁ is one of aryl and heteroaryl, and optionally is substituted with oneor more groups independently selected from halo, hydroxy, carboxy,cyano, nitro, trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl,heteroaryl, aryloxy, heteroaryloxy, and —NR^(a)R^(b);

R₂ is H or methyl;

R₃ is H or methyl;

R₄ is (C₂-C₆)alkyl or (C₂-C₆)alkenyl, which (C₂-C₆)alkyl or(C₂-C₆)alkenyl is optionally substituted with one or more groupsindependently selected from oxo, halo, hydroxy, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy,heteroaryloxy, and —NR^(c)R^(d);

R^(a) and R^(b) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆) alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(a) andR^(b) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino; and

R^(c) and R^(d) are each independently H, (C₁-C₆)alkyl, aryl,heteroaryl, aryl(C₁-C₆) alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(c) andR^(d) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino;

or a salt thereof.

In one specific embodiment the invention provides a compound of formulaI which is:

or a salt thereof.

Processes for preparing compounds of formula I are provided as furtherembodiments of the invention and are illustrated in the followingschemes in which the meanings of the generic radicals are as given aboveunless otherwise qualified. For example, compounds of formula I can beprepared as illustrated in Scheme 2.

In cases where compounds are sufficiently basic or acidic, a salt of acompound of formula I can be useful as an intermediate for isolating orpurifying a compound of formula I. Additionally, administration of acompound of formula I as a pharmaceutically acceptable acid or base saltmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 150 mg/kg, e.g., from about 10 to about 125 mg/kg of bodyweight per day, such as 3 to about 75 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 120 mg/kg/day, mostpreferably in the range of 15 to 90 mg/kg/day.

The compound is conveniently formulated in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form. Inone embodiment, the invention provides a composition comprising acompound of the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The ability of a compound of the invention to inhibit a bacterial RNApolymerase can be determined using biochemical models that are wellknown to the art, or as described in the Examples.

The ability of a compound of the invention to inhibit a bacterial growthin culture can be determined using biochemical models that are wellknown to the art, or as described in the Examples.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Synthesis of4-hydroxy-6-pentyl-3-(3-phenylpropanoyl))-2-pyrone (1) a.4-hydroxy-6-methyl-3-(3-phenylpropanoyl)-2-pyrone

Triethylamine (1.12 ml; 8 mmol; Aldrich) was added to4-hydroxy-6-methyl-2-pyrone (1.01 g; 8 mmol; Aldrich) in 60 ml CH₂Cl₂,and the reaction mixture was stirred under N₂ for 10 min at 0° C. DMAP(293.3 mg; 2.4 mmol; Aldrich) and DCC (1.65 g; 8 mmol; Aldrich) wereadded, and the reaction mixture was stirred under N₂ for 15 min at 0° C.Hydrocinnamic acid (1.20 g; 8 mmol; Aldrich) was added, and the reactionmixture was stirred N₂ for 12 h at room temperature. The reactionmixture was filtered on celite to remove urea, acidified to pH ˜2 byaddition of by 1 N HCl, diluted by addition of 60 ml water was added,and extracted with CH₂Cl₂ (2×100 ml). The organic layer was washed withbrine, dried over anhydrous Na₂SO₄, and evaporated under vacuum. Theproduct was isolated by chromatography on silica gel. Yield: 58%. MS(MALDI): calculated, m/z 259.1 (MH⁺); found, 259.1.

b. 4-hydroxy-6-pentyl-3-(3-phenylpropanoyl)-2-pyrone (1)

1.5 M LDA in cyclohexane (1.34 ml; 2 mmol; Aldrich) was added to4-hydroxy-6-methyl-3-(3-phenylpropanoyl)-2-pyrone (258.27 mg; 1 mmol;Example 1.1) in 8.4 ml anhydrous THF, and the reaction mixture wasstirred under N₂ for 0.5 h at 0° C. 1-iodo-butane (136.8 μl; 1.2 mmol;Aldrich) and HMPA (167 ul; 0.96 mmol: Aldrich) were added sequentiallyunder N₂, and the reaction mixture was stirred under N₂ for 2 h at 0° C.and 5 h at room temperature. The reaction mixture was acidified byaddition of 1 N HCl (16 ml) and extracted with ethyl acetate (3×20 ml).The organic layer was washed with brine, dried over anhydrous Na₂SO₄,and evaporated under vacuum. The product was isolated by chromatographyon silica gel. Yield: 20%. MS (MALDI): calculated, m/z 315.2 (MH⁺);found, 315.1.

Example 2 Assay of Inhibition of Bacterial RNA Polymerase Example 2.1Assay of Inhibition of Escherichia coli RNA Polymerase

Fluorescence-detected RNA polymerase assays with E. coli RNA polymerasewere performed by a modification of the procedure of Kuhlman et al.,2004 [Kuhlman, P., Duff, H. & Galant, A. (2004) A fluorescence-basedassay for multisubunit DNA-dependent RNA polymerases. Anal. Biochem.324, 183-190]. Reaction mixtures contained (20 μl): 0-100 nM testcompound, 75 nM E. coli RNA polymerase σ⁷⁰ holoenzyme, 20 nM 384 bp DNAfragment containing the bacteriophage T4 N25 promoter, 100 μM ATP, 100μM GTP, 100 μM UTP, 100 μM CTP, 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10mM MgCl₂, 1 mM DTT, 10 μg/ml bovine serum albumin, and 5.5% glycerol.Reaction components other than DNA and NTPs were pre-incubated for 10min at 37° C. Reactions were carried out by addition of DNA andincubation for 5 min at 37° C., followed by addition of NTPs andincubation for 60 min at 37° C. DNA was removed by addition of 1 μl 5 mMCaCl₂ and 2 U DNaseI (Ambion, Inc.), followed by incubation for 90 minat 37° C. RNA was quantified by addition of 100 μl RiboGreen RNAQuantitation Reagent (Invitrogen, Inc.; 1:500 dilution in Tris-HCl, pH8.0, 1 mM EDTA), followed by incubation for 10 min at 25° C., followedby measurement of fluorescence intensity [excitation wavelength=485 nmand emission wavelength=535 nm; QuantaMaster QM1 spectrofluorometer(PTI, Inc.)]. IC50 is defined as the concentration of inhibitorresulting in 50% inhibition of RNA polymerase activity. Data arepresented in Table 1.

Example 2.2 Assay of Inhibition of Mycobacterium tuberculosis RNAPolymerase

Fluorescence-detected RNA polymerase assays with M. tuberculosis RNApolymerase were performed as in Example 2.1, using reaction mixturescontaining (20 μl): 0-100 nM test compound, 75 nM M. tuberculosis RNApolymerase core enzyme, 300 nM M. tuberculosis σ ^(A), 20 nM 384 bp DNAfragment containing the bacteriophage T4 N25 promoter, 100 μM ATP, 100μM GTP, 100 μM UTP, 100 μM CTP, 40 mM Tris-HCl, pH 8.0, 80 mM NaCl, 5 mMMgCl₂, 2.5 mM DTT, and 12.7% glycerol. IC50 is defined as theconcentration of inhibitor resulting in 50% inhibition of RNA polymeraseactivity. Data are presented in Table 1.

Example 2.3 Assay of Inhibition of Staphylococcus aureus RNA Polymerase

Fluorescence-detected RNA polymerase assays with S. aureus RNApolymerase were performed as in Example 2.1, using reaction mixturescontaining (20 μl): 0-100 nM test compound, 75 nM S. aureus RNApolymerase core enzyme, 300 nM S. aureus σ ^(A), 20 nM 384 bp DNAfragment containing the bacteriophage T4 N25 promoter, 100 μM ATP, 100μM GTP, 100 μM UTP, 100 μM CTP, 40 mM Tris-HCl, pH 8.0, 80 mM NaCl, 5 mMMgCl₂, 2.5 mM DTT, and 12.7% glycerol. IC50 is defined as theconcentration of inhibitor resulting in 50% inhibition of RNA polymeraseactivity. Data are presented in Table 1.

Example 3 Assay of Inhibition of Bacterial Growth in Culture Example 3.1Assay of Inhibition of Growth of Staphylococcus aureus, EnterococcusFaecalis, Enterococcus faecalis, and Escherichia coli

Minimum inhibitory concentrations (MICs) for Staphylococcus aureus ATCC12600, Staphylococcus aureus ATCC 13709 MSSA, Staphylococcus aureusBAA-1707 MRSA (MW2), Staphylococcus aureus BAA-1717 MRSA (USA300),Enterococcus faecalis ATCC 19433, Enterococcus faecium ATCC19434, andEscherichia coli D21f2tolC were quantified using spiral gradientendpoint assays, essentially as described [Wallace, A. and Corkill, J.(1989) Application of the spiral plating method to study antimicrobialaction. J. Microbiol. Meths. 10, 303-310; Paton, J., Holt, A., andBywater, M. (1990) Measurement of MICs of antibacterial agents by spiralgradient endpoint compared with conventional dilution methods. Int. J.Exp. Gin. Chemother. 3, 31-38; Schalkowsky S. (1994) Measures ofsusceptibility from a spiral gradient of drug concentrations. Adv. Exp.Med. Biol. 349, 107-120]. Assays employed exponential-gradient platescontaining 150 mm×4 mm Mueller-Hinton II cation-adjusted agar and0.4-100 μg/ml of test compound. Plates were prepared using an Autoplate4000 spiral plater (Spiral Biotech, Inc.). Saturated overnight cultureswere swabbed radially onto plates, and plates were incubated for 16 h at37° C. For each culture, the streak length was measured using a clearplastic template (Spiral Biotech, Inc.), the test-compound concentrationat the streak endpoint was calculated using the program SGE (SpiralBiotech, Inc.), and the MIC was defined as the calculated test-compoundconcentration at the streak endpoint. Data are presented in Table 1.

Example 3.2 Assay of Inhibition of Growth of Mycobacterium tuberculosis

MICs for Mycobacterium tuberculosis were quantified using microplateAlamar Blue assays as described [Collins, L. & Franzblau, S. (1997)Microplate Alamar Blue assay versus BACTEC 460 system forhigh-throughput screening of compounds against Mycobacteriumtuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41,1004-1009]. Data are presented in the following Table.

Compound 1 IC50 37 E. coli RNAP (μM) IC50 12 M. tuberculosis RNAP (μM)IC50 40 S. aureus RNAP (μM) MIC 9 M. tuberculosis H37Rv (μg/ml) MIC 6 S.aureus 12600 (μg/ml) MIC 9 S. aureus 13709 (μg/ml) MIC 5 S. aureus MRSA1707 (μg/ml) MIC 8 S. aureus MRSA 1717 (μg/ml) MIC 3 E. faecalis 19433(μg/ml) MIC 7 E. faecium 19434 (μg/ml) MIC 3 E. coli D21f2tolC (μg/ml)

Example 4

The following illustrate representative pharmaceutical dosage forms,containing a compound of formula I (‘Compound X’), for therapeutic orprophylactic use in humans.

(i) Tablet 1 mg/tablet Compound X = 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet Compound X = 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule Compound X = 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml Compound X = (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml Compound X = (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 01N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can Compound X = 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of formula I:

wherein: R₁ is one of aryl and heteroaryl, and optionally is substitutedwith one or more groups independently selected from halo, hydroxy,carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl,heteroaryl, aryloxy, heteroaryloxy, and —NR^(a)R^(b); R₂ is one of H,halo, hydroxy, carboxy, cyano, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy, heteroaryloxy, or—NR^(c)R^(d), wherein any (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, andheteroaryl, and is optionally substituted with one or more halo,hydroxy, carboxy, cyano, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy or —NR^(c)R^(d); R₃ is one of H, halo, hydroxy,carboxy, cyano, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl,heteroaryl, aryloxy, heteroaryloxy, or —NR^(e)R^(f), wherein any(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, and heteroaryl, and isoptionally substituted with one or more halo, hydroxy, carboxy, cyano,nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy or—NR^(e)R^(f); R₄ is (C₂-C₆)alkyl or (C₂-C₆)alkenyl, which (C₂-C₆)alkylor (C₂-C₆)alkenyl is optionally substituted with one or more groupsindependently selected from oxo, halo, hydroxy, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy,heteroaryloxy, and —NR^(g)R^(h); R^(a) and R^(b) are each independentlyH, (C₁-C₆)alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl, orheteroaryl(C₁-C₆) alkyl; or R^(a) and R^(b) together with the nitrogento which they are attached form a morpholino, piperazino, pyrrolidino orpiperidino; R^(c) and R^(d) are each independently H, (C₁-C₆)alkyl,aryl, heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆) alkyl; or R^(c)and R^(d) together with the nitrogen to which they are attached form amorpholino, piperazino, pyrrolidino or piperidino; R^(e) and R^(f) areeach independently H, (C₁-C₆)alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl,or heteroaryl(C₁-C₆) alkyl; or R^(e) and R^(f) together with thenitrogen to which they are attached form a morpholino, piperazino,pyrrolidino or piperidino; and R^(g) and R^(h) are each independently H,(C₁-C₆)alkyl, aryl, heteroaryl, aryl(C₁-C₆)alkyl, or heteroaryl(C₁-C₆)alkyl; or R^(g) and R^(h) together with the nitrogen to which they areattached form a morpholino, piperazino, pyrrolidino or piperidino; or asalt thereof.
 2. The compound of claim 1 wherein R₂ is H or methyl; andR₃ is H or methyl.
 3. The compound of claim 1 wherein R₁ is phenyl whichis optionally substituted with one or more groups independently selectedfrom halo, hydroxy, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkanoyloxy, aryl, heteroaryl, aryloxy, heteroaryloxy, and—NR^(a)R^(b).
 4. The compound of claim 1 wherein R₁ is phenyl.
 5. Thecompound of claim 1 wherein R₄ is n-butyl, or i-pentyl.
 6. The compoundof claim 1 which is:

or a salt thereof.
 7. A composition comprising a compound of formula Ias described in claim 1, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable vehicle.
 8. A method for inhibiting abacterial RNA polymerase comprising contacting the bacterial RNApolymerase with a compound of formula I as described in claim 1, or asalt thereof.
 9. A method for inhibiting the growth of a bacteriumcomprising contacting the bacterium with a compound of formula I asdescribed in claim 1, or a salt thereof.
 10. A method for treating abacterial infection in a mammal comprising administering to the mammalan effective amount of a compound of formula I as described in claim 1,or a pharmaceutically acceptable salt thereof. 11-12. (canceled)
 13. Themethod of claim 10, wherein the bacteria is selected from Mycobacteriumtuberculosis, Staphylococcus aureus MSSA and MRSA, Enterococcusfaecalis, Enterococcus faecium, and Escherichia coli D21f21tolC. 14-15.(canceled)